Mitotic Chromosome Structure: Reproducibility of Folding and Symmetry between Sister Chromatids

Mitotic chromosome structure and pathways of mitotic condensation remain unknown. The limited amount of structural data on mitotic chromosome structure makes it impossible to distinguish between several mutually conflicting models. Here we used a Chinese hamster ovary cell line with three different lac operator-tagged vector insertions distributed over an ∼1 μm chromosome arm region to determine positioning reproducibility, long-range correlation in large-scale chromatin folding, and sister chromatid symmetry in minimally perturbed, metaphase chromosomes. The three-dimensional positions of these lac operator-tagged spots, stained with lac repressor, were measured in isolated metaphase chromosomes relative to the central chromatid axes labeled with antibodies to topoisomerase II. Longitudinal, but not axial, positioning of spots was reproducible but showed intrinsic variability, up to ∼300 nm, between sister chromatids. Spot positions on the same chromatid were uncorrelated, and no correlation or symmetry between the positions of corresponding spots on sister chromatids was detectable, showing the absence of highly ordered, long-range chromatin folding over tens of mega-basepairs. Our observations are in agreement with the absence of any regular, reproducible helical, last level of chromosome folding, but remain consistent with any hierarchical folding model in which irregularity in folding exists at one or multiple levels.     

Modeling epigenome folding: formation and dynamics of topologically associated chromatin domains

Genomes of eukaryotes are partitioned into domains of functionally distinct chromatin states. These domains are stably inherited across many cell generations and can be remodeled in response to developmental and external cues, hence contributing to the robustness and plasticity of expression patterns and cell phenotypes. Remarkably, recent studies indicate that these 1D epigenomic domains tend to fold into 3D topologically associated domains forming specialized nuclear chromatin compartments. However, the general mechanisms behind such compartmentalization including the contribution of epigenetic regulation remain unclear. Here, we address the question of the coupling between chromatin folding and epigenome. Using polymer physics, we analyze the properties of a block copolymer model that accounts for local epigenomic information. Considering copolymers build from the epigenomic landscape of Drosophila, we observe a very good agreement with the folding patterns observed in chromosome conformation capture experiments. Moreover, this model provides a physical basis for the existence of multistability in epigenome folding at sub-chromosomal scale. We show how experiments are fully consistent with multistable conformations where topologically associated domains of the same epigenomic state interact dynamically with each other. Our approach provides a general framework to improve our understanding of chromatin folding during cell cycle and differentiation and its relation to epigenetics.     

Revisiting higher-order and large-scale chromatin organization

The past several years has seen increasing appreciation for plasticity of higher-level chromatin folding. Four distinct '30nm' chromatin fiber structures have been identified, while new in situ imaging approaches have questioned the universality of 30nm chromatin fibers as building blocks for chromosome folding in vivo. 3C-based approaches have provided a non-microscopic, genomic approach to investigating chromosome folding while uncovering a plethora of long-distance cis interactions difficult to accommodate in traditional hierarchical chromatin folding models. Recent microscopy based studies have suggested complex topologies co-existing within linear interphase chromosome structures. These results call for a reappraisal of traditional models of higher-level chromatin folding.     

Chromatin globules: a common motif of higher order chromosome structure?

Recent technological advances in the field of chromosome conformation capture are facilitating tremendous progress in the ability to map the three-dimensional (3D) organization of chromosomes at a resolution of several Kb and at the scale of complete genomes. Here we review progress in analyzing chromosome organization in human cells by building 3D models of chromatin based on comprehensive chromatin interaction datasets. We describe recent experiments that suggest that long-range interactions between active functional elements are sufficient to drive folding of local chromatin domains into compact globular states. We propose that chromatin globules are commonly formed along chromosomes, in a cell type specific pattern, as a result of frequent long-range interactions among active genes and nearby regulatory elements. Further, we speculate that increasingly longer range interactions can drive aggregation of groups of globular domains. This process would yield a compartmentalized chromosome conformation, consistent with recent observations obtained with genome-wide chromatin interaction mapping.     

Gene functioning and storage within a folded genome

In mammals, genomic DNA that is roughly 2 m long is folded to fit the size of the cell nucleus that has a diameter of about 10 μm. The folding of genomic DNA is mediated via assembly of DNA-protein complex, chromatin. In addition to the reduction of genomic DNA linear dimensions, the assembly of chromatin allows to discriminate and to mark active (transcribed) and repressed (non-transcribed) genes. Consequently, epigenetic regulation of gene expression occurs at the level of DNA packaging in chromatin. Taking into account the increasing attention of scientific community toward epigenetic systems of gene regulation, it is very important to understand how DNA folding in chromatin is related to gene activity. For many years the hierarchical model of DNA folding was the most popular. It was assumed that nucleosome fiber (10-nm fiber) is folded into 30-nm fiber and further on into chromatin loops attached to a nuclear/chromosome scaffold. Recent studies have demonstrated that there is much less regularity in chromatin folding within the cell nucleus. The very existence of 30-nm chromatin fibers in living cells was questioned. On the other hand, it was found that chromosomes are partitioned into self-interacting spatial domains that restrict the area of enhancers action. Thus, TADs can be considered as structural-functional domains of the chromosomes. Here we discuss the modern view of DNA packaging within the cell nucleus in relation to the regulation of gene expression. Special attention is paid to the possible mechanisms of the chromatin fiber self-assembly into TADs. We discuss the model postulating that partitioning of the chromosome into TADs is determined by the distribution of active and inactive chromatin segments along the chromosome.This article was specially invited by the editors and represents work by leading researchers.     

From nucleosome to chromosome: a dynamic organization of genetic information

Gene activity is controlled at different levels of chromatin organization, which involve genomic sequences, nucleosome structure, chromatin folding and chromosome arrangement. These levels are interconnected and influence each other. At the basic level nucleosomes generally occlude the DNA sequence from interacting with DNA-binding proteins. Evidently, nucleosome positioning is a major factor in gene control and chromatin organization. Understanding the biological rules that govern the deposition and removal of the nucleosomes to and from the chromatin fiber is the key to understanding gene regulation and chromatin organization. In this review we describe and discuss the relationship between the different levels of chromatin organization in plants and animals.     

Chromatin modifiers and recombination factors promote a telomere fold-back structure,that is lost during replicative senescence

Telomeres have the ability to adopt a lariat conformation and hence, engage in long and short distance intra-chromosome interactions. Budding yeast telomeres were proposed to fold back into subtelomeric regions, but a robust assay to quantitatively characterize this structure has been lacking. Therefore, it is not well understood how the interactions between telomeres and non-telomeric regions are established and regulated. We employ a telomere chromosome conformation capture (Telo-3C) approach to directly analyze telomere folding and its maintenance in S. cerevisiae. We identify the histone modifiers Sir2, Sin3 and Set2 as critical regulators for telomere folding, which suggests that a distinct telomeric chromatin environment is a major requirement for the folding of yeast telomeres. We demonstrate that telomeres are not folded when cells enter replicative senescence, which occurs independently of short telomere length. Indeed, Sir2, Sin3 and Set2 protein levels are decreased during senescence and their absence may thereby prevent telomere folding. Additionally, we show that the homologous recombination machinery, including the Rad51 and Rad52 proteins, as well as the checkpoint component Rad53 are essential for establishing the telomere fold-back structure. This study outlines a method to interrogate telomere-subtelomere interactions at a single unmodified yeast telomere. Using this method, we provide insights into how the spatial arrangement of the chromosome end structure is established and demonstrate that telomere folding is compromised throughout replicative senescence.     

Engineered chromosome regions with altered sequence composition demonstrate hierarchical large-scale folding within metaphase chromosomes

Mitotic chromosome structure and DNA sequence requirements for normal chromosomal condensation remain unknown. We engineered labeled chromosome regions with altered scaffold-associated region (SAR) sequence composition as a formal test of the radial loop and other chromosome models. Chinese hamster ovary cells were isolated containing high density insertions of a transgene containing lac operator repeats and a dihydrofolate reductase gene, with or without flanking SAR sequences. Lac repressor staining provided high resolution labeling with good preservation of chromosome ultrastructure. No evidence emerged for differential targeting of SAR sequences to a chromosome axis within native chromosomes. SAR sequences distributed uniformly throughout the native chromosome cross section and chromosome regions containing a high density of SAR transgene insertions showed normal diameter and folding. Ultrastructural analysis of two different transgene insertion sites, both spanning less than the full chromatin width, clearly contradicted predictions of simple radial loop models while providing strong support for hierarchical models of chromosome architecture. Specifically, an approximately 250-nm-diam folding subunit was visualized directly within fully condensed metaphase chromosomes. Our results contradict predictions of simple radial loop models and provide the first unambiguous demonstration of a hierarchical folding subunit above the level of the 30-nm fiber within normally condensed metaphase chromosomes.     

3D chromatin conformation correlates with replication timing and is conserved in resting cells

Although chromatin folding is known to be of functional importance to control the gene expression program, less is known regarding its interplay with DNA replication. Here, using Circular Chromatin Conformation Capture combined with high-throughput sequencing, we identified megabase-sized self-interacting domains in the nucleus of a human lymphoblastoid cell line, as well as in cycling and resting peripheral blood mononuclear cells (PBMC). Strikingly, the boundaries of those domains coincide with early-initiation zones in every cell types. Preferential interactions have been observed between the consecutive early-initiation zones, but also between those separated by several tens of megabases. Thus, the 3D conformation of chromatin is strongly correlated with the replication timing along the whole chromosome. We furthermore provide direct clues that, in addition to the timing value per se, the shape of the timing profile at a given locus defines its set of genomic contacts. As this timing-related scheme of chromatin organization exists in lymphoblastoid cells, resting and cycling PBMC, this indicates that it is maintained several weeks or months after the previous S-phase. Lastly, our work highlights that the major chromatin changes accompanying PBMC entry into cell cycle occur while keeping largely unchanged the long-range chromatin contacts.     

The three-dimensional structure of in vitro reconstituted <Emphasis Type="Italic">Xenopus laevis</Emphasis> chromosomes by EM tomography

We have studied the in vitro reconstitution of sperm nuclei and small DNA templates to mitotic chromatin in Xenopus laevis egg extracts by three-dimensional (3D) electron microscopy (EM) tomography. Using specifically developed software, the reconstituted chromatin was interpreted in terms of nucleosomal patterns and the overall chromatin connectivity. The condensed chromatin formed from small DNA templates was characterized by aligned arrays of packed nucleosomal clusters having a typical 10-nm spacing between nucleosomes within the same cluster and a 30-nm spacing between nucleosomes in different clusters. A similar short-range nucleosomal clustering was also observed in condensed chromosomes; however, the clusters were smaller, and they were organized in 30- to 40-nm large domains. An analysis of the overall chromatin connectivity in condensed chromosomes showed that the 30–40-nm domains are themselves organized into a regularly spaced and interconnected 3D chromatin network that extends uniformly throughout the chromosomal volume, providing little indication of a systematic large-scale organization. Based on their topology and high degree of interconnectedness, it is unlikely that 30–40-nm domains arise from the folding of local stretches of nucleosomal fibers. Instead, they appear to be formed by the close apposition of more distant chromatin segments. By combining 3D immunolabeling and EM tomography, we found topoisomerase II to be randomly distributed within this network, while the stable maintenance of chromosomes head domain of condensin was preferentially associated with the 30–40-nm chromatin domains. These observations suggest that 30–40-nm domains are essential for establishing long-range chromatin associations that are central for chromosome condensation. Electronic supplementary material The online version of this article (doi ) contains supplementary material, which is available to authorized users.     

The three-dimensional architecture of a bacterial genome and its alteration by genetic perturbation

We have determined the three-dimensional (3D) architecture of the Caulobacter crescentus genome by combining genome-wide chromatin interaction detection, live-cell imaging, and computational modeling. Using chromosome conformation capture carbon copy (5C), we derive ~13 kb resolution 3D models of the Caulobacter genome. The resulting models illustrate that the genome is ellipsoidal with periodically arranged arms. The parS sites, a pair of short contiguous sequence elements known to be involved in chromosome segregation, are positioned at one pole, where they anchor the chromosome to the cell and contribute to the formation of a compact chromatin conformation. Repositioning these elements resulted in rotations of the chromosome that changed the subcellular positions of most genes. Such rotations did not lead to large-scale changes in gene expression, indicating that genome folding does not strongly affect gene regulation. Collectively, our data suggest that genome folding is globally dictated by the parS sites and chromosome segregation.     

Common pathway of chromosome condensation in Mammalian cells

Chromatin folding in the interphase nucleus is not known. We compared the pattern of chromatin condensation in Indian muntjac, Chinese hamster ovary, murine pre B, and K562 human erythroleukemia cells during the cell cycle. Fluorescent microscopy showed that chromosome condensation follows a general pathway. Synchronized cells were reversibly permeabilized and used to isolate interphase chromatin structures. Based on their structures two major categories of intermediates were distinguished: (1) decondensed chromatin and (2) condensed chromosomal forms. (1) Chromatin forms were found between the G1 and mid-S phase involving veil-like, supercoiled, fibrous, ribboned structures; (2) condensing chromosomal forms appeared in the late-S, G2, and M phase, including strings, chromatin bodies, elongated pre-chromosomes, pre-condensed chromosomes, and metaphase chromosomes. Results demonstrate that interphase chromosomes are clustered in domains; condensing interphase chromosomes are linearly arranged. Our results raise questions related to telomer sequences and to the chemical nature of chromosome connectivity.     

Visualization of early chromosome condensation: a hierarchical folding, axial glue model of chromosome structure

Current models of mitotic chromosome structure are based largely on the examination of maximally condensed metaphase chromosomes. Here, we test these models by correlating the distribution of two scaffold components with the appearance of prophase chromosome folding intermediates. We confirm an axial distribution of topoisomerase IIalpha and the condensin subunit, structural maintenance of chromosomes 2 (SMC2), in unextracted metaphase chromosomes, with SMC2 localizing to a 150-200-nm-diameter central core. In contrast to predictions of radial loop/scaffold models, this axial distribution does not appear until late prophase, after formation of uniformly condensed middle prophase chromosomes. Instead, SMC2 associates throughout early and middle prophase chromatids, frequently forming foci over the chromosome exterior. Early prophase condensation occurs through folding of large-scale chromatin fibers into condensed masses. These resolve into linear, 200-300-nm-diameter middle prophase chromatids that double in diameter by late prophase. We propose a unified model of chromosome structure in which hierarchical levels of chromatin folding are stabilized late in mitosis by an axial "glue."     

Cell-cycle-dependent structural transitions in the human CENP-A nucleosome in vivo

In eukaryotes, DNA is packaged into chromatin by canonical histone proteins. The specialized histone H3 variant CENP-A provides an epigenetic and structural basis for chromosome segregation by replacing H3 at centromeres. Unlike exclusively octameric canonical H3 nucleosomes, CENP-A nucleosomes have been shown to exist as octamers, hexamers, and tetramers. An intriguing possibility reconciling these observations is that CENP-A nucleosomes cycle between octamers and tetramers in?vivo. We tested this hypothesis by tracking CENP-A nucleosomal components, structure, chromatin folding, and covalent modifications across the human cell cycle. We report that CENP-A nucleosomes alter from tetramers to octamers before replication and revert to tetramers after replication. These structural transitions are accompanied by reversible chaperone binding, chromatin fiber folding changes, and previously undescribed modifications within the histone fold domains of CENP-A and H4. Our results reveal a cyclical nature to CENP-A nucleosome structure and have implications for the maintenance of epigenetic memory after centromere replication.